Guanine nucleotide dissociation inhibitor (GDI) plays an essential role in the recycling of Rab proteins, small GTPases that regulate membrane vesicle traffic. We have established that Rab recycling involves a Hsp90-GDI chaperone system. The principle focus of this proposal is to understand the biochemical and molecular basis for the function of the Hsp90 chaperone complex in mediating GDI-Rab protein interaction during Rab recycling.We propose 3 specific aims to provide structural, molecular and biochemical insight into the structure/function relationships between GDI, Rab and their sequential physiological protein interactions with components of the Hsp90 chaperone machinery. We will explore the general hypothesis that transfer of Rab from the lipid bilayer to GDI during recycling involves a Hsp90 chaperone complex in an analogous fashion to the manner in which steroid hormone receptors (SHR) complexes are primed for steroid hormone binding by the Hsp90 chaperone complex. Specific Aim 1 will continue to expand our structural studies to determine the molecular basis of GDI function. These studies will include determining the structure of beta and delta GDI isoforms, determining the structure of the native alpha GDI-Rab3A-GG complex isolated from bovine brain, and determining structure(s) of alpha GDI-Hsp90 chaperone complexes. Specific Aim 2 will apply biochemical approaches to identify and characterize the basis for the interaction of GDI with components of the Hsp90 chaperone complex. These studies will take advantage of the extensive knowledge of the role of the Hsp90 chaperone complexes in regulation of SHR and signaling kinase pathways. Specific Aim 3 will take a molecular approach to identify the basis for protein interactions between GDI, Rab and the Hsp90 chaperone complex. These three aims will contribute significantly to advancing our basic understanding of the general role of GDI and Rab GTPases in membrane transport, an area of high interest to contemporary cell biology. They will provide insight into a broad spectrum of diseases given the role of GDI and Rab in mental retardation, diseases of hematopoietic lineage, eye disease and cell proliferation (cancer) related to changes in Hsp90 regulated signaling pathways.